Project Summary This proposal develops a strategy to expand the chemical space that is accessible to biology by engineering enzymes to build new amino acids from non-natural, synthetic precursors. As one of the fundamental building blocks of life, amino acids are incorporated into diagnostic tools and human medicines, such as proteins, antibiotics, and anticancer drugs. Although alterations to a chemical structure can have a profound and positive effect on a compound?s usefulness, biosynthetic pathways have access to limited chemical diversity compared to the vast array of functional groups employed by medicinal chemists. Research in this proposal aims to develop new enzymes that can convert cheap, commercially-available building blocks into non-canonical amino acids. This pursuit will require creating entirely new enzyme functions, which is a major challenge in enzymology. We will collect structural and mechanistic information that will to help understand how new enzymes work and guide our engineering strategies. These enzymes will have activities that shortcut traditional synthetic methodology, work without the need of toxic solvents and reagents, and are fully biodegradable. Incorporating these new enzymes into a living host will enable the biosynthesis of diverse non-canonical amino acids in vivo, which can subsequently be elaborated into proteins, peptides, and small molecules with new and improved functionality.